Voltage-gated K + channels contribute significantly to the regulation of resting and action potentials of classically excitable cells such as neurons and muscle cells. The K + flux required for membrane potential control is regulated by the state of different gates existing in the channels, which underlines the necessity for their biophysical characterization. Although conformational changes in the voltage sensor, the activation gate (A-gate) and different inactivation gates (N-type inactivation, slow-inactivation (P-type and C-type)) have been studied rather extensively in isolation, the way in which the movements of these different gates and sensors are energetically and kinetically coupled is less understood. In Aim 1, of the current research proposal, we will determine the state of the A-gate during entry and recovery from P-type inactivation. To assess the state of the A-gate in inactivated (i.e. non-conducting) channels we will apply experimental strategies based on gated access of intracellularly applied cysteine modifying agents and intracellular blockers to areas in the conduction pathway located behind the A-gate. Cysteines introduced into specific positions in the sixth transraembrane segment of Shaker K + channels. The extent of cysteine modification or blocker trapping will be determined from the reduction of ionic currents in inside-out patches. The chemicals will be applied for short durations in different states of the channel (open, fully inactivated) using rapid solution exchange. The .coupling between the A-gate and the P-gate will be analyzed as the probability of A-gate opening as a function of voltage, time, and duration of exposure to reagent/blocker. Using similar approaches, the effect of known modulators of slow inactivation (permeant ions, mutations is the pore region and the sixth transmembrane segment) will be analyzed to reveal the coupling between the A- and P-gates (Aim 2).